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研究生:王惠宗
研究生(外文):Hui-Tsung Wang
論文名稱:光觸媒二氧化鈦奈米材質抗菌活性之探討
論文名稱(外文):The antimicrobial activity of TiO2 nano-photocatalyst
指導教授:蔡國珍
指導教授(外文):Guo-Jane Tsai
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:103
中文關鍵詞:光觸媒二氧化鈦抗菌活性
外文關鍵詞:PhotocatalystTitanium dioxideAntimicrobial activity
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本論文以光觸媒TiO2奈米粒(OPT,~ 30nm)及鉑修飾TiO2奈米粒(SONANO,~ 35nm)為測試物質,首先建立其抗菌活性的分析條件,並分析其對不同細菌,包含6株G(+)與7株G(-)細菌,以及3株真菌的抗菌活性。其次探討此二種TiO2所產生的活性氧物質;最後探討溫度、溶氧量、pH、鹽度、菌齡以及微量食品成分等不同因子對TiO2抗菌活性的影響。

紫外光(366 nm)及日光燈(13 W)下作用10小時後,SONANO對次甲基藍的分解率分別為57.73%及36.41%,OPT則為37.36%及9.00%。抗菌測試以10公分光照距離、2小時光照時間、0.01%(w / v)之SONANO添加濃度,103 cfu / mL之E. coli起始濃度以及0.2公分之E. coli菌懸液深度,可得到較佳的抗菌效果。在紫外光照射下,除了Lactobacillus acidophilus及Listeria monocytogenes外,OPT對包括Bacillus cereus、Clostridium perfringens、Staphylococcus aureus、Staphylococcus aureus(MRSA)在內之四株常見G(+)細菌,以及Aeromonas hydrophila、Escherichia coli、Escherichia coli(O157)、Pseudomonas aeruginosa、Pseudomonas fluorescens、Salmonella choleraesuis、Vibrio parahaemolyticus等七株G(-)細菌作用2小時後,各菌殘存率在12.31% ~ 33.86%,SONANO則在16.82% ~ 59.71%。至於OPT在紫外光照射下對Saccharomyces cerevisiae、Penicillium minioluteum及Trichophyton mentagrophytes三株真菌作用2小時後,三株真菌殘存率在16.22% ~ 29.39%,SONANO為19.03% ~ 66.96%。在日光燈照射下,OPT幾乎無抗菌活性,SONANO可使前述四株G(+)及七株G(-)病原菌之殘存率降至4.57% ~ 46.67%,三株測試真菌之殘存率為40.85% ~ 66.99%。OPT與SONANO在紫外光照射下均會產生超氧陰離子和氫氧自由基;SONANO經由紫外光或日光燈照射,2 小時後產生約0.4 mg / L之H2O2,OPT經由紫外光照射則產生約0.33 mg / L之H2O2;當添加H2O2濃度大於0.3 mg / L後,影響E. coli以及S. aureus殘存率情形和光觸媒TiO2所產生的抗菌活性相似。5℃到45℃測試溫度不影響OPT抗菌活性,SONANO對E. coli抗菌活性則隨溫度升高而上升。pH 4.5 ~ pH 9.0測試範圍不影響OPT抗菌活性,SONANO以在pH 6.5 ~ pH 7.0之抗菌活性最大,pH上升或下降均會降低其活性。4.20 ~ 29.57 ppm之菌懸液溶氧量不會影響SONANO抗菌活性。鹽度(NaCl)增高會降低OPT和SONANO的抗菌活性。E. coli菌齡會影響其對OPT和SONANO的耐受性,以對數生長期末期之E. coli菌體最敏感。添加bovine serium albumin和glucose後,會降低SONANO的抗菌活性,但添加500 ppm之soybean oil反而增加其抗菌活性,可能與soybean oil在光觸媒作用下所產生的脂質過氧化物malondialdehyde(MDA)有關;分析SONANO與500 ppm之soybean oil在紫外光和日光燈下照射20分鐘,分別有3.29 ppm和2.65 ppm之MDA產生,可提高光觸媒SONANO的抗菌效果。
The types of nano-photocatalyst, TiO2 (OPT, 30 nm) and Platinum modified TiO2 (SONANO, 35 nm) were used. Thirteen commonly found pathogen including 6 G(+) bacteria and 7 G(-) bacteria, and 3 fungal species were used to the tested microorganisms. The aim of this study was to investigate the antimicrobial activity of OPT and SONANO with UV and fluorescent lamp illumination. The production of reactive oxygen species from OPT and SONANO, and the effects of some factors including temperature, dissolved oxygen, pH, NaCl and bacterial ages on the antimicrobial activity of these two photocatalysts were also studied.

The degradation rates of methylene blue for SONANO by UV (366 nm) and fluorescent lamp (13 W) for 10 hours were 57.73% and 36.41%. Respectively, the degradation rates for OPT were 37.36% and 9.00%. The following conductions were set out to obtain higher antimicrobial activity: cell suspension with a density of 103 cfu / mL, and 0.2 cm in depth were added with 0.01% OPT or SONANO, and illuminated by UV or fluorescent lamp for 2 hours, which was 10 cm above the surface of cell suspension. Except Lactobacillus acidophilus and Listeria monocytogenes, both of which were more resistant to OPT and SONANO, the survival ratio of 11 stains of the tested bacteria in OPT and SONANO by UV illumination were 12.31% ~ 33.86% and 16.82% ~ 59.71%, respectively. The survival ratio of 3 fungal species for OPT and SONANO by UV illumination were 16.22% ~ 29.39% and 19.03% ~ 66.96%, respectively. The survival ratio of 11 bacteria and 3 fungal species in SONANO by fluorescent lamp illumination were 4.57% ~ 46.67%, and 40.85% ~ 66.99%, respectively. The OPT had little antimicrobial activity by fluorescent lamp illumination. OPT and SONANO could produce superoxide anions, hydroxyl radicals and H2O2 during irradiation with UV. SONANO produced about 0.4 mg/L H2O2 illuminated by UV or fluorescent lamp for 2 hours, and OPT produced about 0.33 mg/L H2O2 illuminated by UV for 2 hours. The survival ratio for E. coli and S. aureus obtained from 0.3 mg/L pure H2O2 and from OPT/SONANO were similar. Temperature in the range of 5℃ to 45℃ does not affect the antimicrobial activity of OPT. However, the survival ratio of E. coli in SONANO by UV or fluorescent lamp decreases with the increasing temperature from 5℃ to 45℃. Dissolved oxygen (DO) in the range of 4.20 to 29.57 ppm has little effect on the antimicrobial activity of SONANO. SONANO has the highest antimicrobial activity at pH 6.5 ~ pH 7.0; while the pH does not affect OPT. The cell age of E. coli affects to susceptibility to OPT/SONANO, with the cell in late-log phase being most sensitive. The presence of NaCl, bovine serum albumin and glucose decrease the antimicrobial activity of SONANO. However, 500 ppm soybean oil can increase the antimicrobial activity of SONANO. This may be due to the production of malondialdehyde (MDA) from oil oxidation that was promoted by SONANO. The amounts of MDA produced from SONANO with oxygenized soybean oil by UV and fluorescent lamp illumination for 20 min were 3.29 ppm and 2.65 ppm, respectively.
壹、前言……………………………………………………….……. 1
貳、文獻整理………………………………………………….……. 3
1. 光觸媒TiO2概述...….………………………………….……... 3
1.1. 觸媒簡介………………………………………………...... 3
1.2. 光觸媒TiO2特性………………………..………………... 3
1.3 光觸媒TiO2研究歷史……………..……………...…….… 5
1.4 光觸媒TiO2氧化還原原理……………………….…….… 6
1.5 光觸媒TiO2反應機制……..………………………...….… 6
1.6 超氧陰離子、氫氧自由基與過氧化氫的產生……...….... 8
1.7影響光觸媒光催化效率的因數……………………...……. 9
1.7.1添加劑的影響…………...…………………....…...…..... 9
1.7.2光強度的影響…………………………………...……... 11
1.7.3溶氧量的影響………………………………...………... 12
1.7.4 pH值的影響………………………………...………..... 13
1.7.5溼度的影響………………………………….................. 14
2. 光觸媒TiO2對細菌的影響………………………………....... 15
2.1 光觸媒TiO2 添加濃度對細菌的作用…...……..……...... 15
2.2光觸媒TiO2對不同細菌之抗菌效果………...………...... 16
2.3光觸媒TiO2對不同細菌菌數的作用………...………...... 16
2.4光觸媒TiO2包覆在器皿上對細菌的作用………....…..... 17
2.5光觸媒TiO2對細胞膜與細胞壁的作用………….…….... 18
2.6光觸媒TiO2對毒素的作用………………………….….... 20
2.7光觸媒TiO2抑制或殺滅細菌的可能模式……...……….. 21
參、材料設備與實驗方法……………………………..………….. 27
1. 實驗流程……………………..………………………………. 27
2. 實驗材料與設備………………………..……………………. 28
2.1光觸媒TiO2………………………..……………..………. 28
2.2 菌株………………………………..………..……………. 28
2.3 培養基……………………………..…..…………………. 28
2.4 化學藥品…………..……………..………………………. 29
2.5 器材及設備.………………………………………..…….. 30
3. 實驗方法……………………………………………….…….. 31
3.1光觸媒TiO2抗菌活性分析之操作建立………….…..….. 31
3.1.1 光觸媒TiO2奈米粒懸浮液製備……………….....….. 31
3.1.2 以掃描式電子顯微鏡(scanning electron microscope,
SEM)觀察光觸媒TiO2粒徑大小…….…………….. 31
3.1.3. 光觸媒TiO2分解次甲基藍(methylene blue)之活性
探討………………………..…………….…………….. 31
3.1.4不同光源強度(高度)影響…………….….…......….. 32
3.1.5不同反應時間之影響………………..…….….......….. 32
3.1.6 SONANO添加濃度和起始菌數之交互影響…....….. 33
3.1.7不同菌液液面深度之影響……………..…..….….….. 33
3.2光觸媒TiO2抗菌效果與抗菌機制探討…..….…….….….. 33
3.2.1細菌與真菌抗菌活性分析…..….…….……….….….. 33
3.2.2 細菌catalase活性分析…..….…..………….….……. 34
3.2.3細菌孢子抗菌活性分析…………………….….…….. 35
3.2.3.1 細菌孢子製備…………………..……….….…….. 35
3.2.3.2 對孢子之抗菌測試.…….……..……….….…..….. 36
3.2.4 微生物培養及菌數分析方法……..….….….……….. 36
3.2.4.1 微生物培養方法……..….……………….……….. 36
3.2.4.2 菌數分析方法……..…….……………….……….. 36
3.2.5 超氧陰離子〔•O2-〕之分析………………...…….. 37
3.2.6 氫氧自由基〔•OH-〕之分析……………….…….. 37
3.2.7過氧化氫H2O2之分析……………….…………...….. 38
3.3 不同因數對光觸媒TiO2抗菌活性之影響….….….....….. 38
3.3.1 溫度……………………………..……….…….…...….. 38
3.3.2 溶氧量…………………………..………..………...….. 39
3.3.3 pH值…………………...………..….….…………...….. 39
3.3.4鹽度………………….....……...……….…………...….. 39
3.3.5菌齡………………….....……...……….…………...….. 40
3.3.6微量食品成分………......……..……….…………...….. 40
3.3.7脂質過氧化產物丙二醛(malondialdehyde, MDA)分析…….…....…………………………...…….………….. 40
肆、結果與討論……………………………………...…………….. 42
1. 光觸媒TiO2抗菌活性分析之操作建立…………….……….. 42
1.1 TiO2奈米粒懸浮液粒徑觀察…………………….....…….. 42
1.2 光觸媒TiO2分解次甲基藍(methylene blue)之活性探討42
1.3 不同光源強度(高度)影響…………………….....…….. 43
1.4 不同反應時間之影響…………………………………….. 44
1.5 SONANO添加濃度和起始菌數之交互影響…….………. 47
1.6 不同菌液液面深度之影響…………………...…….…….. 50
2. 光觸媒TiO2抗菌效果與抗菌機制探討……….……….…….. 51
2.1 細菌、真菌與細菌孢子抗菌活性分析…………….…….. 51
2.1.1細菌與真菌抗菌活性分析…………………...….…….. 51
2.1.2細菌孢子抗菌活性分析……………………...….…….. 54
2.2 超氧陰離子〔•O2-〕之分析………..…….…………….. 54
2.3 氫氧自由基〔•OH-〕之分析…………….…………….. 55
2.4 過氧化氫H2O2之分析………………..…….…………….. 56
3. 不同因數對光觸媒TiO2抗菌活性之影響…………………… 58
3.1 溫度………………...……………………………………... 58
3.2 溶氧量……………………………...……………………... 60
3.3 pH值……………………………………...…………....….. 62
3.4 鹽度………………………………...………………….….. 65
3.5 菌齡………………………………………..…….……….. 66
3.6 微量食品成分……………………………..……….…….. 67
伍、結論…………………………………………………….……... 70
陸、參考文獻………………………………………………..…….. 73

圖表目錄
圖一、光觸媒TiO2光催化反應簡圖………………………….…….. 8
圖二、以SEM觀察未經超音波處理之光觸媒TiO2………….…... 86
圖三、以SEM觀察經超音波處理後之奈米光觸媒TiO2..…...…... 87
圖四、紫外光和日光燈照射下,0.01% OPT及SONANO對次甲
基藍的分解能力………………….…..……..…..………….. 88
圖五、0.01% SONANO在不同光源高度照射下,於2小時後對
E. coli殘存率的影響…………………...…………….…….. 88
圖六、0.01% SONANO在不同光照時間下對E. coli殘存率之影
響……………………………...……………...……...…..….. 89
圖七、0.01% SONANO對不同起始菌數之E. coli懸浮液,經紫
外光與日光燈照射2小時後的殘存率影響情形……...…... 89
圖八、0.01% SONANO對不同菌量之E. coli懸浮液經紫外光與
日光燈照射2小時後之菌體死滅係數……………..……... 90
圖九、不同濃度SONANO經不同光源照射2小時後,對不同起
始菌濃度之E. coli殘存率影響……...……………...…….. 91
圖十、不同濃度SONANO經紫外光照射2小時後,對不同起始
菌濃度之E. coli菌體死滅係數影響...…………………….. 92
圖十一、0.01% SONANO經紫外光與日光燈照射2小時後對不
同菌液液體深度之E. coli殘存率影響……….......…..….. 92
圖十二、不同菌株在菌濃度為103 cfu / mL時的catalase活性,與
0.01% OPT和SONANO經紫外光與日光燈照射2小時
後E. coli懸浮液殘存率的相關情形……………....…..... 93
圖十三、以紫外光和日光燈分別照射0.01% OPT與SONANO懸
浮液後,所產生〔.O2-〕之變化情形………………....….. 94
圖十四、以紫外光和日光燈分別照射0.01% OPT與SONANO懸
浮液後,所產生〔.OH-〕之變化情形………………...….. 94
圖十五、以紫外光和日光燈分別照射0.01% OPT與SONANO懸
浮液後,所產生H2O2之情形……….....………………..... 95
圖十六、不同濃度之H2O2於2小時後對E. coli和S. aureus殘存
率之影響……….....…………………..………….………. 95
圖十七、0.01% OPT與SONANO在不同反應溫度下經紫外光與
日光燈照射2小時後,對E. coli殘存率之影響……….... 96
圖十八、0.01% SONANO經紫外光與日光燈照射2小時後,對不
同溶氧量之E. coli懸浮液殘存率之影響..……….…….. 96
圖十九、0.01% OPT與SONANO經紫外光與日光燈照射2小時
後,對不同pH值之E. coli懸浮液殘存率之影響…...….. 97
圖二十、以紫外光和日光燈分別照射0.01% OPT與SONANO懸
浮液後, pH之變化情形………….……..……...……... 97
圖二十一、0.01% OPT與SONANO經紫外光與日光燈照射2
小時後,對不同鹽度之E. coli懸浮液殘存率之影響... 98
圖二十二、0.01%OPT與SONANO經紫外光與日光燈照射2小
時後,對不同菌齡之E. coli懸浮液殘存率之影響…. 98
圖二十三、0.01% SONANO經紫外光與日光燈照射2小時後,
對不同微量食品成分添加之E. coli 懸浮液殘存率的影
響情形…………………………………………………. 99
圖二十四、0.01% SONANO經紫外光與日光燈照射2小時後,
對不同濃度malondialdehyde(MDA)添加之E. coli懸
浮液殘存率的影響情形……………………….…….. 100
圖二十五、0.01% SONANO在紫外光與日光燈照射下,對soybean
oil氧化後產生MDA之影響……………..……..….. 100
表一、測試的菌株、來源、培養溫度與適合之培養基……..……. 28
表二、0.01% OPT和SONANO經紫外光與日光燈照射後,對不
同菌懸液殘存率的影響………………………………...… 101
表三、0.01% OPT和SONANO經紫外光與日光燈照射,對不同
菌懸液在2小時、4小時、6小時後殘存率的影響…....… 102
表四、0.01% OPT和SONANO經紫外光與日光燈照射,對不同
細菌孢子懸浮液在2小時、4小時、6小時後殘存率的影響103
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